Induction of broadly neutralizing antibodies (bnAbs) is a critical unmet goal of HIV vaccine development. BnAb DH511 is of high interest as a vaccine lead due to its very high breadth and the high in vivo protective potency of MPER bNAbs. Key challenges for inducing DH511-like bnAbs are: (a) the low affinity of DH511- like precursors for HIV peptides and proteins, (b) the restriction on bnAb angle of approach imposed by the recessed, membrane-proximal epitope environment and (c) the absence of the DH511 epitope from most soluble, native-like trimers. A promising strategy to initiate DH511-like bnAb induction is germline targeting, in which suitable DH511-class precursors are specifically activated using engineered immunogens, thus selecting BCRs with the potential to develop broad neutralization in the absence of autoreactivity. This approach will also help circumvent steric problems associated with the recessed location of the epitope, by priming precursors with known genetic and structural potential to mature into bnAbs compatible with MPER steric restraints. In this project, which is Project 1 of a multi-project collaborative proposal, we will engineer epitope-scaffold immunogens that bind with high affinity to and activate DH511-like precursors, using computational design and directed evolution. As known bnAbs are highly mutated, vaccine induction of bnAbs following a germline-targeting prime will likely require sequential immunization with other immunogens designed to shepherd affinity maturation of the B-cell receptor. We will develop different classes of boosting immunogens, including epitope- scaffolds with more native epitopes, membrane-protein scaffolds and membrane-bound Env variants stabilized in a conformation to which DH511 binds strongly. Structural studies of soluble and membrane- bound immunogens in complex with DH511 lineage members will guide immunogen development. The Animal Core of this collaborative proposal will generate knock-in mice that express DH511-like precursors, and Project 2 will use those mice to test B cell priming and boosting in vivo. Project 2 will conduct sequential prime/boost immunization experiments in knock-in mice and use ELISA, cytometry, single B cell sorting and sequencing and neutralization assays to track and optimize affinity maturation, providing experimental feedback to Project 1 to allow for iterative improvement of immunogens. In summary, these studies seek to develop novel HIV vaccine candidates and also to shift HIV vaccine research towards a reductionist approach based on state-of-the-art protein engineering to develop germline-targeting and boosting immunogens, development of human Ig knock-in mouse models to enable testing of human-repertoire-specific vaccines, and in-depth analysis of vaccine-induced affinity maturation pathways in vivo to guide iterative vaccine optimization.